Antisense Oligonucleotides Targeting Jagged 1 Reduce House Dust Mite-induced Goblet Cell Metaplasia in the Adult Murine Lung.

Goblet cell metaplasia, excessive mucus production, and inadequate mucus clearance accompany and exacerbate multiple chronic respiratory disorders, such as asthma and chronic obstructive pulmonary disease. Notch signaling plays a central role in controlling the fate of multiple cell types in the lung, including goblet cells. In the present study, we explored the therapeutic potential of modulating the Notch pathway in the adult murine lung using chemically modified antisense oligonucleotides (ASOs). To this end, we designed and characterized ASOs targeting the Notch receptors Notch1, Notch2, and Notch3 and the Notch ligands Jag1 (Jagged 1) and Jag2 (Jagged 2). Pulmonary delivery of ASOs in healthy mice or mice exposed to house dust mite, a commonly used mouse model of asthma, resulted in a significant reduction of the respective mRNAs in the lung. Furthermore, ASO-mediated knockdown of Jag1 or Notch2 in the lungs of healthy adult mice led to the downregulation of the club cell marker Scgb1a1 and the concomitant upregulation of the ciliated cell marker FoxJ1 (forkhead box J1). Similarly, ASO-mediated knockdown of Jag1 or Notch2 in the house dust mite disease model led to reduced goblet cell metaplasia and decreased mucus production. Because goblet cell metaplasia and excessive mucus secretion are a common basis for many lung pathologies, we propose that ASO-mediated inhibition of JAG1 could provide a novel therapeutic path for the treatment of multiple chronic respiratory diseases.

Directed RNase H Cleavage of Nascent Transcripts Causes Transcription Termination.

An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.

A modular analysis of microglia gene expression, insights into the aged phenotype.

BACKGROUND: Microglia are multifunctional cells that are key players in brain development and homeostasis. Recent years have seen tremendous growth in our understanding of the role microglia play in neurodegeneration, CNS injury, and developmental disorders. Given that microglia show diverse functional phenotypes, there is a need for more precise tools to characterize microglial states. Here, we experimentally define gene modules as the foundation for describing microglial functional states. RESULTS: In an effort to develop a comprehensive classification scheme, we profiled transcriptomes of mouse microglia in a stimulus panel with 96 different conditions. Using the transcriptomic data, we generated fine-resolution gene modules that are robustly preserved across datasets. These modules served as the basis for a combinatorial code that we then used to characterize microglial activation under various inflammatory stimulus conditions. CONCLUSIONS: The microglial gene modules described here were robustly preserved, and could be applied to in vivo as well as in vitro conditions to dissociate the signaling pathways that distinguish acutely inflamed microglia from aged microglia. The microglial gene modules presented here are a novel resource for classifying and characterizing microglial states in health and disease.

Antisense suppression of the nonsense mediated decay factor Upf3b as a potential treatment for diseases caused by nonsense mutations.

BACKGROUND: About 11% of all human genetic diseases are caused by nonsense mutations that generate premature translation termination codons (PTCs) in messenger RNAs (mRNA). PTCs not only lead to the production of truncated proteins, but also often result in  decreased mRNA abundance due to  nonsense-mediated mRNA decay (NMD). Although pharmacological inhibition of NMD could be an attractive therapeutic approach for the treatment of diseases caused by nonsense mutations, NMD also regulates the expression of 10-20% of the normal transcriptome. RESULTS: Here, we investigate whether NMD can be inhibited to stabilize mutant mRNAs, which may subsequently produce functional proteins, without having a major impact on the normal transcriptome. We develop antisense oligonucleotides (ASOs) to systematically deplete each component in the NMD pathway. We find that ASO-mediated depletion of each NMD factor elicits different magnitudes of NMD inhibition in vitro and are differentially tolerated in normal mice. Among all of the NMD factors, Upf3b depletion is well tolerated, consistent with previous reports that UPF3B is not essential for development and regulates only a subset of the endogenous NMD substrates. While minimally impacting the normal transcriptome, Upf3b-ASO treatment significantly stabilizes the PTC-containing dystrophin mRNA in mdx mice and coagulation factor IX mRNA in a hemophilia mouse model. Furthermore, when combined with reagents promoting translational read-through, Upf3b-ASO treatment leads to the production of functional factor IX protein in hemophilia mice. CONCLUSIONS: These data demonstrate that ASO-mediated reduction of the NMD factor Upf3b could be an effective and safe approach for the treatment of diseases caused by nonsense mutations.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.

Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment.

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.

Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function.

Viable constitutive and tamoxifen inducible liver-specific RNase H1 knockout mice that expressed no RNase H1 activity in hepatocytes showed increased R-loop levels and reduced mitochondrial encoded DNA and mRNA levels, suggesting impaired mitochondrial R-loop processing, transcription and mitochondrial DNA replication. These changes resulted in mitochondrial dysfunction with marked changes in mitochondrial fusion, fission, morphology and transcriptional changes reflective of mitochondrial damage and stress. Liver degeneration ensued, as indicated by apoptosis, fibrosis and increased transaminase levels. Antisense oligonucleotides (ASOs) designed to serve as substrates for RNase H1 were inactive in the hepatocytes from the RNase H1 knockout mice and in vivo, demonstrating that RNase H1 is necessary for the activity of DNA-like ASOs. During liver regeneration, a clone of hepatocytes that expressed RNase H1 developed and partially restored mitochondrial and liver function.

Partial Hepatectomy Induced Long Noncoding RNA Inhibits Hepatocyte Proliferation during Liver Regeneration.

Liver regeneration after partial hepatectomy (PHx) is a complex and well-orchestrated biological process in which synchronized cell proliferation is induced in response to the loss of liver mass. To define long noncoding RNAs (lncRNAs) that participate in the regulation of liver regeneration, we performed microarray analysis and identified more than 400 lncRNAs exhibiting significantly altered expression. Of these, one lncRNA, LncPHx2 (Long noncoding RNA induced by PHx 2), was highly upregulated during liver regeneration. Depletion of LncPHx2 during liver regeneration using antisense oligonucleotides led to a transient increase in hepatocyte proliferation and more rapid liver regeneration. Gene expression analysis showed that LncPHx2 depletion resulted in upregulation of mRNAs encoding proteins known to promote cell proliferation, including MCM components, DNA polymerases, histone proteins, and transcription factors. LncPHx2 interacts with the mRNAs of MCM components, making it a candidate to regulate the expression of MCMs and other genes post-transcriptionally. Collectively, our data demonstrate that LncPHx2 is a key lncRNA that participates in a negative feedback loop modulating hepatocyte proliferation through RNA-RNA interactions.

Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1.

The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program.

GATA-3 dose-dependent checkpoints in early T cell commitment.

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.

Surgery for tracheobronchomalacia.

Tracheobronchomalacia (TBM) refers to a weakening of the anterior tracheal rings leading to splaying and collapse of the central airways. In this report, we review the treatment of TBM, including preoperative workup, intraoperative anesthesia management, and surgical technique for posterior splinting tracheobronchoplasty. Imperative in the preoperative preparation is a stent trial in which an airway stent is placed to temporarily relieve the TBM and reassess for improvement in symptoms. Definitive therapy is then carried out with posterior splinting tracheoplasty or tracheobronchoplasty. Surgical results are generally excellent with the majority of patients having significant improvements in breathing.

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45.

Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.

Synthetic in vivo validation of gene network circuitry.

Embryonic development is controlled by networks of interacting regulatory genes. The individual linkages of gene regulatory networks (GRNs) are customarily validated by functional cis-regulatory analysis, but an additional approach to validation is to rewire GRN circuitry to test experimentally predictions derived from network structure. Here we use this synthetic method to challenge specific predictions of the sea urchin embryo endomesoderm GRN. Expression vectors generated by in vitro recombination of exogenous sequences into BACs were used to cause elements of a nonskeletogenic mesoderm GRN to be deployed in skeletogenic cells and to detect their effects. The result of reengineering the regulatory circuitry in this way was to divert the developmental program of these cells from skeletogenesis to pigment cell formation, confirming a direct prediction of the GRN. In addition, the experiment revealed previously undetected cryptic repression functions.

Proteome and system ontology of hemorrhagic shock: exploring early constitutive changes in postshock mesenteric lymph.

BACKGROUND: Postshock mesenteric lymph (PSML) is the mechanistic link between splanchnic ischemia reperfusion (IR) and remote organ injury. We hypothesize that an unbiased inspection of the proteome of PSML will reveal previously unrecognized aberrations in systems biology provoked by hemorrhage-induced mesenteric IR injury in vivo. METHODS: Shock was induced in male Sprague-Dawley rats by controlled hemorrhage, and the mesenteric duct was cannulated for lymph collection. Preshock and postshock lymph were collected for differential in-gel electrophoresis (DIGE)-based proteomics. Proteins that increased or decreased in relative concentration > or =1.5-fold were selected for trypsin digestion and analysis by mass spectrometry (MS). RESULTS: Evidence of tissue injury was detected by an increase in cell/tissue proteins in PSML. Components of coagulation were depleted, whereas products of hemolysis were increased. Haptoglobin was decreased, which supports an early postshock hemolytic process. Interestingly, several protective protease inhibitors were decreased in PSML. The unexpected findings were an increase in alpha-enolase (a key glycolitic enzyme and cell-surface plasminogen binding receptor, +2.4-fold change) and increased major urinary protein (MUP, a sex-specific lipid-binding protein, +17.1-fold change) in PSML. CONCLUSION: A proteomic evaluation of PSML revealed evidence of several shock-associated processes: protein release from tissue injury, depletion of coagulation factors and evidence of hemolysis, depletion of protective protease inhibitors, and an increase in abundance of lipid carriers. These results suggest that constitutive changes in the proteome of PSML may provide novel insights into the complex pathophysiology of postshock systems biology.

HMGB1 is markedly elevated within 6 hours of mechanical trauma in humans.

High-mobility group box 1 (HMGB1) is a late mediator of the systemic inflammation associated with sepsis. Recently, HMGB1 has been shown in animals to be a mediator of hemorrhage-induced organ dysfunction. However, the time course of plasma HMGB1 elevations after trauma in humans remains to be elucidated. Consequently, we hypothesized that mechanical trauma in humans would result in early significant elevations of plasma HMGB1. Trauma patients at risk for multiple organ failure (ISS > or = 15) were identified for inclusion (n = 23), and postinjury plasma samples were assayed for HMGB1 by enzyme-linked immunosorbent assay. Comparison of postinjury HMGB1 levels with markers for patient outcome (age, injury severity score, units of red blood cell (RBC) transfused per first 24 h, and base deficit) was performed. To investigate whether postinjury transfusion contributes to elevations of circulating HMGB1, levels were determined in both leuko-reduced and non-leuko-reduced packed RBCs. Plasma HMGB1 was elevated more than 30-fold above healthy controls within 1 h of injury (median, 57.76 vs. 1.77 ng/mL; P < 0.003), peaked from 2 to 6 h postinjury (median, 526.18 ng/mL; P < 0.01 vs. control), and remained elevated above control through 136 h. No clear relationship was evident between postinjury HMGB1 levels and markers for patient outcome. High-mobility group box 1 levels increase with duration of RBC storage, although concentrations did not account for postinjury plasma levels. Leuko-reduced attenuated HMGB1 levels in packed RBCs by approximately 55% (P < 0.01). Plasma HMGB1 is significantly increased within 1 h of trauma in humans with marked elevations occurring from 2 to 6 h postinjury. These results suggest that, in contrast to sepsis, HMGB1 release is an early event after traumatic injury in humans. Thus, HMGB1 may be integral to the early inflammatory response to trauma and is a potential target for future therapeutics.

Hemoglobin-based oxygen carrier induces heme oxygenase-1 in the heart and lung but not brain.

BACKGROUND: The clinical sequel of ischemia and reperfusion remains a challenge in several clinical areas. Overexpression of heme oxygenase-1 (HO-1), using viral vectors, endotoxemia, and hypoxia, provides protection against ischemia and reperfusion injury. To date, however, no clinically viable therapy exists to safely induce HO-1. We have recently observed that administration of a hemoglobin-based oxygen carrier (HBOC) attenuates postinjury systemic inflammation. We have further demonstrated that an HBOC can induce HO-1 in vitro. We now explore the tissue-specific induction of heme oxygenase-1 after administration of an HBOC. STUDY DESIGN: Rats were infused with doses of HBOC or saline through femoral vein injection (n=5 per group). Animals were sacrificed and organs were flushed. Heart, lung, and brain samples were taken for evaluation of total organ levels of HO-1 induction and for histologic localization of the cellular expression of the HO-1. Heat shock protein 72 levels were also analyzed to determine whether HO-1 induction was a generalized stress response. RESULTS: Both the heart and lung demonstrated a dose-dependent induction of total organ HO-1. Interestingly, brain tissue did not have any significant amount of HO-1, either at baseline or after HBOC therapy. The cellular localization of HO-1 between organs was also specific, predominantly occurring in the cardiac myocyte and alveolar macrophages. Heat shock protein 72 levels were not significantly changed in any group examined, suggesting the induction of HO-1 is specific. CONCLUSIONS: This study demonstrates that a clinically accessible product, HBOC, can specifically and selectively induce the expression of the protective enzyme HO-1 in vivo. These findings begin to characterize which organ systems may benefit by preischemic treatments with HBOC and further expand potential clinical applications of HBOCs.

Heme oxygenase-1 induction in macrophages by a hemoglobin-based oxygen carrier reduces endotoxin-stimulated cytokine secretion.

The inflammatory response after an insult may provoke further tissue damage, and the macrophage is central in this pathophysiology. Induction of heme oxygenase-1 (HO-1) attenuates postshock organ dysfunction, although the mechanism remains unclear. We hypothesized that HO-1 induction modifies the cytokine profile of LPS-stimulated macrophages. Heme oxygenase-1 was induced in murine and human macrophages with varying concentrations of a hemoglobin-based oxygen carrier (HBOC). Heme oxygenase-1 expression was analyzed by Western blotting of whole cell lysates. Macrophages were pretreated with HBOC for 4 h, then media with LPS were added for up to 24 h. The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) was used to inhibit the effects of HO-1. Supernatants were analyzed for IL-6, IL-10, TNF-alpha, and monocyte chemotactic protein 1 (MCP-1) by enzyme-linked immunosorbent assay. Incubation of cells with HBOC produced a dose-dependent expression of HO-1. Heme oxygenase-1 expression decreased LPS-stimulated secretion of MCP-1, IL-6, IL-10, and TNF-alpha at both 4 and 24 h in murine and human macrophages. The addition of ZnPP to inhibit HO-1 partially restored MCP-1 and IL-6 secretion in murine macrophages. Furthermore, immunofluorescent microscopy revealed HBOC-induced HO-1 inhibited LPS-stimulated nuclear translocation of the p65 subunit of nuclear factor-kappaB. In summary, HBOC incubation of macrophages induced HO-1 expression, which reduced LPS-mediated cytokine release, and that MCP-1 and IL-6 secretion could be partially restored with ZnPP. These data encourage continued investigation into the role of HO-1 in protecting against posttraumatic organ dysfunction and the clinical potential of HBOC for HO-1 induction.

Arachidonic acid in postshock mesenteric lymph induces pulmonary synthesis of leukotriene B4.

Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remains elusive. Previous work has shown that administration of a phospholipase A(2) (PLA(2)) inhibitor attenuated postshock ALI and also identified a non-ionic lipid within the postshock mesenteric lymph (PSML) responsible for polymorphonuclear neutrophil (PMN) priming. Consequently, we hypothesized that gut-derived leukotriene B(4) (LTB(4)) is a key mediator in the pathogenesis of ALI. Trauma/hemorrhagic shock (T/HS) was induced in male Sprague-Dawley rats and the mesenteric duct cannulated for lymph collection/diversion. PSML, arachidonic acid (AA), and a LTB(4) receptor antagonist were added to PMNs in vitro. LC/MS/MS was employed to identify bioactive lipids in PSML and the lungs. T/HS increased AA in PSML and increased LTB(4) and PMNs in the lung. Lymph diversion decreased lung LTB(4) by 75% and PMNs by 40%. PSML stimulated PMN priming (11.56 +/- 1.25 vs. 3.95 +/- 0.29 nmol O(2)(-)/min; 3.75 x 10(5) cells/ml; P < 0.01) that was attenuated by LTB(4) receptor blockade (2.64 +/- 0.58; P < 0.01). AA stimulated PMNs to produce LTB(4), and AA-induced PMN priming was attenuated by LTB(4) receptor antagonism. Collectively, these data indicate that splanchnic ischemia/reperfusion activates gut PLA(2)-mediated release of AA into the lymph where it is delivered to the lungs, provoking LTB(4) production and subsequent PMN-mediated lung injury.

Postshock mesenteric lymph induces endothelial NF-kappaB activation.

BACKGROUND: Posthemorrhagic shock mesenteric lymph (PSML) has been shown to activate pulmonary endothelial cells and cause lung injury. Although multiple mediators may be involved, most of these effects are mediated by nuclear factor-kappa B (NF-kappaB) activation. Degradation of the inhibitor of kappa B (IkappaB) is a key regulatory step in the activation of NF-kappaB. We therefore hypothesized that PSML would cause IkappaB degradation with subsequent NF-kappaB phosphorylation and nuclear translocation. METHODS: Mesenteric lymph was collected from male rats before shock and each hour after shock for up to 3 h (n = 5). Buffer (control), buffer + 10% (v/v) lymph, or buffer + tumor necrosis factor (10 ng/mL) were incubated with human pulmonary endothelial cells for 30 min and then lysed. Immunoblots of lysates were probed for IkappaB and phospho-p65. Immunohistochemistry was performed on cells grown on glass slides and then treated as above with the third PSML sample. Cells were fixed and then probed for p65. Statistical analysis was performed with Student's t-test and analysis of variance with significance was set at P < 0.05. RESULTS: Western blots of cell lysates for IkappaB demonstrated a steady decrease in total IkappaB with each lymph sample. Phosphorylation of NF-kappaB , p65 component, steadily increased with each PSML sample, with a maximum reached during the third PSML sample, which also significantly increased translocation of NF-kappaB to the nucleus. CONCLUSION: Postshock mesenteric lymph bioactivity is mediated by pathways which involved IkappaB degradation. These pathways offer novel off targets for clinical intervention to prevent the distal organ injury caused by postinjury hemorrhagic shock.

Gelsolin is depleted in post-shock mesenteric lymph.

BACKGROUND: Gelsolin is a plasma protein that functions to depolymerize actin filaments preventing capillary plug formation following tissue injury. It also functions to mediate the inflammatory response by binding proinflammatory lipids such as lysophosphatidic acid, sphingosine-1-phosphate and phosphoinositides. Clinically, reduced gelsolin concentrations have been associated with increased mortality in critically ill, trauma, and burn patients. We have previously shown that following hemorrhagic shock with splanchnic hypoperfusion, mesenteric lymph contains lipid components that cause neutrophil and EC activation and that protein concentrations are severely diluted due to resuscitation. We hypothesized that lipid binding proteins such as gelsolin may be depleted after trauma/hemorrhagic shock leading to increased lipid bioactivity. METHODS: Shock was induced in SD rats by controlled hemorrhage and the mesenteric duct cannulated for lymph collection. Resuscitation was performed by infusing 2x SB volume in NS over 30 min, followed by 1/2 SB volume over 30 min, then 2x SB volume in NS over 60 min. Pre and post-shock lymph was loaded at equal protein concentrations on 2D-gels, followed by trypsin digestion and identification with mass spectrometry (MS-MS). Proteomics data were confirmed with Western blotting then quantitated by densitometry. Analysis of variance was used evaluate statistical data. RESULTS: Gelsolin decreased in mesenteric lymph following hemorrhagic shock. CONCLUSIONS: Gelsolin is found at high levels (comparable to plasma) in mesenteric lymph. Following hemorrhagic shock, gelsolin levels decrease significantly, possibly due to consumption by the actin scavenging system. The magnitude of this change in concentration could release lipid bioactivity and predispose the lung and other organs to capillary injury.